Enhanced adoptive cell therapy

ABSTRACT

The present invention relates to the fields of life sciences and medicine. Specifically, the invention relates to cancer therapies of humans. More specifically, the present invention relates to oncolytic adenoviral vectors alone or together with therapeutic compositions for therapeutic uses and therapeutic methods for cancer. In one aspect the present invention relates to separate administration of adoptive cell therapeutic composition and oncolytic adenoviral vectors. Furthermore, the present invention relates to a pharmaceutical kit and a pharmaceutical composition, both utilizing oncolytic adenoviral vectors.

PRIORITY

This application claims priority of the Finnish national patentapplication number 20135387 filed on Apr. 18, 2013, the contents ofwhich are incorporated herein by reference in entirety.

SEQUENCE LISTING

This patent application contains sequence listing, which is provided inelectronic format and in portable document format. The contents of thesesubmissions are identical.

COLOR DRAWINGS

This patent application contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIELD OF THE INVENTION

The present invention relates to the fields of life sciences andmedicine. Specifically, the invention relates to cancer therapies ofhumans. More specifically, the present invention relates to oncolyticadenoviral vectors alone or together with therapeutic compositions fortherapeutic uses and therapeutic methods for cancer. In one aspect thepresent invention relates to separate administration of adoptive celltherapeutic composition and oncolytic adenoviral vectors. Furthermore,the present invention relates to a pharmaceutical kit and apharmaceutical composition, both utilizing oncolytic adenoviral vectors.

BACKGROUND OF THE INVENTION

Novel therapies are constantly developed for cancer treatment. Adoptivecell therapies (ACT) are a potent approach for treating cancer but alsofor treating other diseases such as infections and graft versus hostdisease. Adoptive cell transfer is the passive transfer of ex vivo growncells, most commonly immune-derived cells, into a host with the goal oftransferring the immunologic functionality and characteristics of thetransplant. Adoptive cell transfer can be autologous, as is common inadoptive T-cell therapies, or allogeneic as typical for treatment ofinfections or graft-versus-host disease. Clinically, common embodimentsof this approach include transfer of either immune-promoting ortolerogenic cells such as lymphocytes to patients to either enhanceimmunity against viruses and cancer or to promote tolerance in thesetting of autoimmune disease, such as type I diabetes or rheumatoidarthritis.

With regard to cancer therapy, the ACT approach was conceived in the1980s by a small number of groups working in the US, one of the leadinggroup being Steven Rosenberg and colleagues working at the NCI. Theadoptive transfer of autologous tumor infiltrating lymphocytes (TILs) orgenetically re-directed peripheral blood mononuclear cells has been usedto successfully treat patients with advanced solid tumors such asmelanoma as well as patients with CD19-expressing hematologicmalignancies. In ACT, the most commonly used cell types are the T-cells,sometimes sorted for CD8+, but other variations include CD4+ cells,NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral bloodmononuclear cells. Cells can be unmodified such as in TIL therapy orgenetically modified. There are two common ways to achieve genetictargeting of T-cells to tumor specific targets. One is transfer of aT-cell receptor with known specificity (TCR therapy) and with matchedhuman leukocyte antigen (HLA, known as major histocompatibility complexin rodents) type. The other is modification of cells with artificialmolecules such as chimeric antigen receptors (CAR). This approach is notdependent on HLA and is more flexible with regard to targetingmolecules. For example, single chain antibodies can be used and CARs canalso incorporate co-stimulatory domains. However, the targets of CARcells need to be on the membrane of target cells, while TCRmodifications can utilize intracellular targets.

For the first decade of ACT development, the focus was on TILs. TILs arefound in tumors, suggesting that tumors trigger an immune response inthe host. This so-called tumor immunogenicity is mediated by tumorantigens. These antigens distinguish the tumor from healthy cells,thereby providing an immunological stimulus.

For example, US2003194804 A1 describes a method for enhancing thereactivity of a T cell toward a tumor cell by utilizing TILs. InUS2003194804 A1 the T cells are exposed to an agent and re-introducinginto the patient. The agent is capable of reducing or preventingexpression or interaction of an endogenous Notch or Notch ligand in theT cell.

U.S. Pat. No. 5,126,132 A describes a method of treating cancer, whereinan effective amount of autologous TILs and a cytokine are used.

Diaz R M et al. (Cancer Res. 2007 Mar. 15; 67(6):2840-8) describe anincrease of the circulating levels of tumor antigen-specific T cells byusing adoptive T cell transfer therapy in combination with vesicularstomatitis virus intratumoral virotherapy. Diaz et al. used OT1 cellsi.e. an artificial monoclonal cell line in adoptive T cell transfertherapy.

While even in early trials of ACTs there were dramatic examples oftreatment benefits, and even cures, most patients did not benefit andmany patients experienced severe side effects. During the first twodecades of adoptive cell therapy, safety of cell transfer per se wasgenerally good, but significant toxicities and even mortality wasassociated with the concomitant treatments used to enhance the therapy,including preconditioning chemotherapy and radiation, and the IL-2 usedafter transfer. Preconditioning is used to kill suppressive cells suchas regulatory T-cells and myeloid derived suppressors in the host, tomodulate the tumor microenvironment and to “make room” for the graft.IL2 is used post-transfer to reduce anergy of the graft and to propagateit.

With regard to efficacy, room is left for improvement. Increasedspecificity and sufficient tumor killing ability of cell therapies ingeneral are warranted. In particular, in the ACT of the prior art thetransferred cells fail to traffic to tumors, and even if they do, theyoften quickly become anergic, are otherwise unable to kill tumor cellsor fail to propagate resulting in a rapid decline of cell numbers.Furthermore, cancers frequently down-regulate human leukocyte antigen(HLA)—known as major histocompatibility complex in animals—in tumorcells, thus resulting in inability of T-cells to kill, as HLA isrequired for presentation of tumor epitopes to the T-cell receptor.

The present invention provides efficient tools and methods for cancertherapeutics utilizing adoptive cell transfers.

BRIEF DESCRIPTION OF THE INVENTION

An object of the present invention is to provide simple methods andtools for overcoming the above problems of inefficient, unsafe andunpredictable cancer therapies. More specifically, the inventionprovides novel methods and means for cell therapy. The objects of theinvention are achieved by viral vectors, methods and arrangements, whichare characterized by what is stated in the independent claims. Thespecific embodiments of the invention are disclosed in the dependentclaims.

The present application describes construction of recombinant viralvectors, methods related to the viral vectors, and their use in tumorcells lines, animal models and cancer patients. The invention is basedon the idea of combining oncolytic adenoviral vectors coding forcytokines or adenoviral vectors with adoptive cell therapeutics forcancer treatment in a novel and inventive way. The invention is based onsurprising effects, i.e. the following improvements in adoptive T-celltherapy: i) recruitment of transferred cells to the tumor, ii)propagation of transferred cells at the tumor, iii) enhanced reactivityof transferred cells at the tumor (FIG. 20). Indeed, the saidcombination of viral vectors and cytokines with adoptive celltherapeutics provides more effective results on wider targets than couldhave been assumed. Effects of the said combination of viral vectorscomprising cytokine transgene with adoptive cell transfer aresynergistic compared to the effects of only viral vectors comprisingcytokine transgene or only adoptive cell transfers. It is a furtherobject of the present invention to provide a combination of tumorinfiltrating lymphocytes (TIL) and transgenic (produced from a virallydelivered transgene) interleukin-2 (IL-2) for the treatment ofmalignancy in humans. The above and various other objects and advantagesof the present invention are achieved by a method of treating malignancyin humans, comprising administering an effective amount of TIL and IL-2,with or without preconditioning chemotherapy and/or radiotherapy, to apatient afflicted with cancer to cause regression or stabilization ofthe cancer.

The present invention relates to a method of treating cancer in asubject, wherein the method comprises separate administration ofadoptive cell therapeutic composition and oncolytic (=replicationcompetent in tumor but not normal cells) adenoviral vectors coding forat least one cytokine to a subject.

The present invention further relates to an oncolytic adenoviral vectorcoding for at least one cytokine together with separate adoptive celltherapeutic composition for use in treatment of cancer.

The present invention further relates to a use of an oncolyticadenoviral vector coding for at least one cytokine together withseparate adoptive cell therapeutic composition in the manufacture of amedicament for treating cancer in a subject.

The present invention also relates to an oncolytic adenoviral vector foruse in increasing the efficacy of adoptive cell therapy or T-celltherapy in a subject.

Also, the present invention relates to a use of an oncolytic adenoviralvector in the manufacture of a medicament for increasing the efficacy ofT-cell therapy in a subject.

Also, the present invention relates to a method of increasing theefficacy of adoptive cell therapy or T-cell therapy in a subject byadministering an oncolytic adenoviral vector to a subject in needthereof.

The present invention also relates to a pharmaceutical kit comprising anadoptive cell therapeutic composition and oncolytic adenoviral vectorscoding for at least one cytokine, wherein the adoptive cell therapeuticcomposition is formulated in a first formulation and the oncolyticadenoviral vectors coding for at least one cytokine are formulated in asecond formulation.

Furthermore, the present invention relates to an oncolytic adenoviralvector comprising

1) an adenovirus serotype 5 (Ad5) nucleic acid backbone comprising a 5/3chimeric fiber knob:

2) E2F1 promoter for tumor specific expression of E1A;

3) a 24 bp deletion (D24) in the Rb binding constant region 2 ofadenoviral E1;

4) a nucleic acid sequence deletion of viral gp19k and 6.7k readingframes; and

5) a nucleic acid sequence encoding at least one cytokine transgene inthe place of the deleted gp19k/6.7K in the E3 region resulting inreplication-associated control of transgene expression under the viralE3 promoter, wherein the cytokine is selected from a group consisting ofinterferon alpha, interferon beta, interferon gamma, complement C5a,IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12,CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18,CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24,CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5(=RANTES), CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8,CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13,CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL1, CXCL6, CXCL7, CXCL8,CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.

Furthermore, the present invention relates to a serotype 3 (Ad3)oncolytic adenoviral vector comprising: a deletion in the E3 area and atumor specific promoter for expression of a transgene in the place ofthe deleted area of E3.

Still, the present invention relates to a pharmaceutical compositioncomprising an oncolytic vector of the invention.

Also, the present invention relates to a method of treating cancer in asubject, wherein the method comprises administration of the oncolyticadenoviral vector of the present invention to a subject in need thereof.Also, the present invention relates to an oncolytic adenoviral vector ofthe present invention for use in treatment of cancer.

Also, the present invention relates to a use of an oncolytic adenoviralvector of the present invention in the manufacture of a medicament fortreating cancer in a subject.

The advantages of the arrangements of the present invention are enhancedtherapeutic effect and reduced side effects. Severe adverse events, evendeaths are prevented, because enhancements in efficacy, and theanti-suppressive effects of our approach, may reduce the need forpreconditioning chemotherapy and/or radiation used in the prior artmethods to “make room” for transferred cells and reduce tumorimmunosuppression. Also, severe adverse events, even deaths areprevented, because separate addition of IL2 used in the prior artmethods to propagate and sustain transferred cells after transferringthem into a patient is not needed if the virus produces it whilereplicating in the tumor. Local production at the tumor can also enhancethe sought-after effects of IL-2 (stimulation and propagation of thegraft) while reducing systemic exposure which is the cause of adverseevents. The present invention provides selective treatments, with lesstoxicity or damage to healthy tissues.

Also, the present invention provides surprising therapeutic effects by:i) Providing trafficking signals to the tumor for example by injectingthe virus vectors comprising recombinant cytokines into tumor. Virusinjection results in production of cytokines relevant for this effect(in reaction to the virus binding to pathogen associated molecularpattern recognition receptors), but much higher effects can be achievedby additional production of the most relevant cytokine as a transgenefrom the virus. ii) Reducing tolerance by increasing danger signals.Virus injection per se can achieve this by binding to pathogenassociated molecular pattern recognition receptors, but the effect canbe enhanced by additional production of a cytokine as a transgene fromthe virus. iii) Inducing HLA expression. Virus infection increases HLAexpression, since cells attempt to present viral epitopes for mountingan anti-viral T-cell response. Unexpectedly, this can be used to enhanceT-cell therapy against tumor epitopes, which requires HLA to work. Theeffect of the virus on HLA is mediated in part by cytokines; productionof said cytokine from the virus can thus induce HLA expression also innearby tumor cells in a surprising embodiment of the invention. iv)Inducing propagation of cells by lifting immunosuppression, mediated byboth the presence of the virus per se (again through the pathogenassociated molecular pattern recognition receptors), but enhanced byproduction of cytokines (FIG. 48). Thus, this approach can solve thecritical obstacles currently hindering adaptive cell therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail bymeans of specific embodiments with reference to the attached drawings,in which

FIG. 1 shows that treatment with Ad5/3 chimeric oncolytic adenovirusincreased cytokine and chemokine secretion in B16-OVA tumors.Interferon-γ can upregulate the expression of HLA (=MHC) class I, thusgenerating a tumor cell phenotype that can effectively be recognized byTILs. Various IFN-γ inducible chemokines (such as RANTES, MIP-1α andMCP-1) are involved in immune cell recruitment, which might promote TILactivation and proliferation. Also trafficking of TILs can be enhancedby up-regulation of these chemokines.

FIG. 2 shows tumor growth control following multiple injections of 5/3chimeric oncolytic adenovirus with or without adoptive cell transfer.Adenovirus treatment alone (A) had little effect on B16-OVA tumor growthcompared to PBS treatment. Adoptive transfer of 500 000 (B) or 2 000 000(C) tumor-specific OT-I lymphocytes in combination with virus injectionsresulted in significant tumor growth control. The poor therapeuticeffect on tumor growth using adenovirus alone or OT-I cells incombination with PBS highlights the major shortcomings of oncolyticvirus and adoptive cell transfer therapies used as single agents,supporting the purpose of the invention to enhance efficacy of adoptivecell therapy using adenovirus.

FIG. 3 shows that adenovirus injections induce T cell trafficking intotumors, and increase proliferation of adoptively transferred T cells intumors. A) Amount of adoptively transferred CD8+ CFSE+ cells increasedin tumors and decreased in blood, draining lymph nodes and spleen of Adtreated mice at day 1 post-treatment, purportedly due to lymphocytetrafficking. The overall CD8+ T-cell count remained high in theadenovirus treated tumors throughout the experiment, suggestingresistance of these cells to the deleterious tumor microenvironmentand/or increased proliferation of the CD8+ T cells. B) On days 6 and 14the OT-I cell proliferation was enhanced in the Ad treated tumors whencompared to the PBS group, seen as a greater fraction of cells that haveundergone cell division (moved toward M7) than in the PBS group.Consequently, treatment with oncolytic adenovirus induces traffickingand proliferation of adoptively transferred TILs. C) Example on howgating of CFSE-positive cells was done. MO indicates cells that have notdivided, M7 shows cells that have divided enough to dilute CFSE to belowdetectable limits (more than 7 times).

FIG. 4 reveals data from humans treated with oncolytic adenovirus ondays indicated by arrows. Virus injection into tumor resulted in adecrease of lymphocytes in blood, reflecting their trafficking totumors.

FIG. 5 reveals data that oncolytic adenovirus injection into a tumor ofa human cancer patient caused influx of CD8+ T cells. Intratumoralinjection of oncolytic adenovirus results in accumulation of CD8+T-cells at the tumor assessed from needle biopsies before and aftertreatment.

FIG. 6 shows results of adenovirus injections combined with adoptivetransfer of T cells. Mice with subcutaneous B16-Ova tumors wereadoptively transferred with 5×10⁵ OT1 lymphocytes intraperitoneally andtumors were left untreated, or injected with PBS or Ad5/3 (see ExamplesMaterials and methods). In an immunosuppressive B16-Ova model similar tohuman melanoma, adoptive transfer of anti-Ova OT1 cells does little.Adding virus injections increases efficacy dramatically (a). CD8+T-cells increase (b). These cells are not anti-Ova T-cells (c).

FIG. 7 reveals dramatic increase in the number of “natural” anti-tumorT-cells due to adoptive transfer and virus injection. Mice withsubcutaneous B16-Ova tumors were adoptively transferred with 5×10⁵ OT1lymphocytes intraperitoneally and tumors were left untreated, orinjected with PBS or Ad5/3 (see Examples Materials and methods).Adoptive transfer+virus injections acts act as catalyst for propagationof “other” T-cells at tumor and local lymph nodes. (a) Trp2 CD8+ cellsat the tumor site. (b) Anti-gp100 CD8+ cell at the tumor site.

FIG. 8 shows activated CD8+ cells in tumor and TIM-3 expression in thetumor on day 14. Mice with subcutaneous B16-Ova tumors were adoptivelytransferred with 5×10⁵ OT1 lymphocytes intraperitoneally and tumors wereleft untreated, or injected with PBS or Ad5/3 (see Examples Materialsand methods). Immunosuppression in immunotherapy: increase in T-cellnumber is not enough if immunosuppression is not removed. There are moreactivated T-cells in virus treated tumors and less immunosuppression.FIG. 9 shows that increase in anti-tumor T-cells and reduction ofimmunosuppression results in systemic immunity against tumor antigens.Mice with subcutaneous B16-Ova tumors were adoptively transferred with5×10⁵ OT1 (a) or 2×10⁶ (b) OT1 lymphocytes intraperitoneally and tumorswere left untreated, or injected with PBS or Ad5/3 (see ExamplesMaterials and methods). Antigen presentation is enhanced by virus:T-cells work better. Systemic immunity against several tumor epitopesresults. (a) expression of co-stimulatory molecules on dendritic cells(CD11c+ CD80+ CD86+) in the tumor on day 14. (b)

IFNg ELISPOT with splenocytes on day 14.

FIG. 10 shows distribution of OTI T-cells following virus injection:trend for trafficking but not enough to explain efficacy. (a) diagram,(b) animal model, (c) tumor.

FIG. 11 reveals that lifting immunosuppression can induce propagation ofcells. Adenovirus treated tumors contained more tumor specificlymphocytes (OT-I cells). In PBS treated tumors OTI cells had arrestedin M5 phase (left arrow), while in the Ad group they continued toproliferate (right arrow).

FIG. 12 shows efficacy of recombinant cytokines (no virus) incombination with OT1 cells.

FIG. 13 shows antitumor efficacy of cytokine-armed adenoviruses combinedwith adoptive T-cell transfer. C57BL/6 mice bearing subcutaneous B16-OVAmelanoma tumors were treated with 1.5×10e6 CD8+ enriched OT-1 T-cellsinteraperitoneally on Day 1. Cytokine-coding adenoviruses or controlvirus Ad5-Luc1 were injected intratumorally on Day 1 and weeklythereafter (1×10e9 viral particles per tumor). Tumor volume wascalculated as previously described (Bramante et al. Serotype chimericoncolytic adenovirus coding for GM-CSF for treatment of sarcoma inrodents and humans. Int J Cancer. 2013 Dec. 24) and tumor sizes areindicated as percentage respective to Day 1, which was set as 100%.Number at risk figure: Number of animals remaining in each experimentalgroup at a given timepoint. Animals were humanely sacrificed when thetumors had exceeded the maximum acceptable size or when any signs ofpain or distress were evident.

FIG. 14 shows effects of different viruses on tumor size.

FIG. 15 shows excellent results of adenoviral vectors comprising mTNFatransgene in combination with OT1 T-cells on reducing the tumor size.

FIG. 16 shows excellent results of adenoviral vectors comprising mIL3transgene in combination with OT1 T-cells on reducing the tumor size.

FIG. 17 shows a schematic of C5a or TNF-α expressing oncolyticadenoviruses. Shown are some important features of the viruses,including the site where the transgenes are inserted.

FIG. 18 shows expression of TNF-α by oncolytic adenovirus in A549 cells.Cells were infected with 10 VP/cell, media was collected at indicatedtime points and ELISA was used to assess the amount of TNF-α in themedia. Virus induces expression and secretion of TNF-α from infectedcells.

FIG. 19 shows biological activity of TNF-alpha produced by oncolyticTNF-alpha-armed oncolytic adenovirus. In this assay, supernatant frominfected cells was used to challenge TNF-sensitive WEHI-13VAR cells,corroborating that oncolytic adenovirus drives expression of functionalcytokines.

FIG. 20 shows dose-dependent killing of human cancer cells by oncolyticadenovirus. As expected, in TNF-alpha insensitive oncolysis permissivehuman A549 or PC3 tumor cells, no difference was observed betweenunarmed control virus and TNF-alpha-expressing oncolytic adenovirus, asmere oncolysis was sufficient to kill cells. However, because humanTNF-alpha is partially active in mouse cells, which are not permissiveto oncolysis by human adenovirus, TNF-alpha contributed to the strongercytotoxicity of the virus seen in B16-OVA mouse cells compared tounarmed virus. Replication-defective virus shows negligible cell-killingcapacity.

FIG. 21 shows that radiation therapy synergizes with TNF-alphaexpressing virus. A) A schematic of the treatment schedule in thisexperiment. Radiation (XRT) was whole body irradiation at a dose of2×2Gy and virus was 1×10⁸ VP/tumor, where each nude mouse carried twoA549 xenografts. B) TNF-alpha virus harbors greater anti-tumor potencythan the unarmed parental virus. Because replication-defective (RD)virus did not kill A549 cells in culture (FIG. 20), the anti-tumoreffect afforded by RD virus in vivo is likely due to innate immuneresponses, including cytokines, NK cells and macrophages, elicited byvirus injections. C) TNF-alpha-expressing virus causes greateranti-tumor effects when combined with clinically relevant doses ofexternal beam irradiation, supporting the clinical translatability ofcytokine-armed viruses. Importantly, these and previous experimentsindicate that TNF-alpha-expressing oncolytic adenovirus is capable ofreplicating and killing cells, arguing that TNF-alpha does not exertantiviral effects against adenovirus.

FIG. 22 shows anti-tumor efficacy of hTNF-alpha-encoding adenovirus onB16-OVA tumors. This experiment is analogous to the one depicted in FIG.26 with C5a virus, demonstrating that TNF-alpha-expression confersgreater therapeutic advantage compared to unarmed virus.

FIG. 23 shows enhanced induction/expansion of tumor-specific CD8+ Tcells in tumors treated with cytokine armed virus (II). Tumors in theexperiment depicted in FIG. 20 were excised and processed for flowcytometric analysis, similar to as in Experiment 11. A greaterinduction/number of OVA-specific CD8+ T cells was detected in tumorstreated with the TNF-encoding virus compared to unarmed control virus,suggesting together with C5a data that rationally selected cytokinesexpressed by oncolytic adenovirus together with the virus-inducedinflammation make a unique tumor milieu that strongly supports expansionand activation of tumor-specific T cells—by inferral and comparison toFIGS. 2B,C also of adoptively transferred T cells.

FIG. 24 shows expression of C5a in A549 cells. Cells were infected with10 VP/cell, media was collected at indicated time points and ELISA wasused to assess the amount of C5a in the media. Results of two individualexperiments are shown.

FIG. 25 shows results of an in vitro chemotaxis assay. The amount ofTHP1 human monocytes passing through a semi-permeable membrane into thelower chamber, as attracted by chemokines in the test supernatants, wasquantified as per manufacturer's instructions (Millipore QCM kit).C5a-expressing virus elicits stronger chemoattractive factors frominfected cells than control virus. Results argue in favor of usingcytokine-armed virus rather than unarmed virus.

FIG. 26 shows anti-tumor efficacy of AdD24-05a in vivo. Establishedsubcutaneous tumors were injected on day 0, 2 and 4 with 1×10⁹ VP ofeach virus or with 50 ul PBS and tumor volumes were measured by caliper.C5a-expressing virus affords superior tumor control compared to controlvirus. As adenovirus does not replicate in or kill mouse cells, i.e. itis non-cytolytic in this model (Young AM et al. Mol Ther. 2012September; 20(9):1676-88, PMID: 22735379), these results underscore therobust ability of the cytokine-armed virus to enhance immunologicalanti-tumor effects, strongly supporting the concept of using it toenhance efficacy of adoptive cell therapy.

FIG. 27 shows enhanced induction/expansion of tumor-specific CD8+ Tcells in tumors treated with cytokine armed virus (I). Tumors inexperiment depicted in FIG. 26 were excised and processed for flowcytometric analysis. Single cell suspensions were stained with antibodyagainst CD8 and with pentamer against anti-ova TCR. C5a-expression bynon-cytolytic adenovirus induces greater OVA-specific CD8 T cell numbersin tumors compared to control viruses, unarmed adenovirus or virusexpressing C5a antagonist, supporting the use of cytokine-armed virus toincrease numbers of adoptively transferred T cells in tumors. Also seeexperiment 13.

FIG. 28 shows a schematic of the adaptive T-cell response.

FIG. 29 shows that adaptively transferred T-cells act as a catalyst(“spark”) for pre-existing T-cells.

FIG. 30 shows that adaptive “spark” results in increase in “natural”anti-tumor T-cells.

FIG. 31 shows the method of adoptive cell transfer.

FIG. 32 shows that with TILT technology of the present invention, toxicpreconditioning (chemo+radiation) and post-conditioning (systemic IL2)can be avoided. (For mechanisms of TILT technology see FIG. 48.)

FIG. 33 shows a schematic of the new virus constructs expressing asingle cytokine. The virus backbone is human adenovirus serotype 5,apart from the fiber knob, which is from serotype 3. Both single anddouble transgenes are under transcriptional control of the virus E3promoter. Both transgenes are placed into the E3 region which is deletedfor gp19k and 6.7k. The E1A protein is deleted for 24 amino acids(“D24”), in constant region 2, rendering Rb binding defective. E1Aexpression is under regulation of the E2F promoter. Some virus generegions are shown for reference.

FIG. 34 shows a schematic of the new virus constructs expressing twocytokines. In one version, ‘ribosome shunt site’/‘ribosome skippingsite’/‘cis-acting hydrolase element’ (CHYSEL) is placed as in-framefusions between each cytokine. The cytokine inserts will be synthesizedas a single polyprotein that is co-translationally cleaved to yield bothcytokines, resulting in addition of several additional amino acids atthe 3′ end of the first cytokine, and a single proline at the 5′ of thelatter cytokine, IL2). In another version, an IRES element separates thetwo cytokines, resulting in synthesis of cytokines with no additionalamino acids.

FIG. 35 shows nucleotide and amino acid sequences of 2A.

FIG. 36 shows TILT Biotherapeutics intravenous adenovirus deliverytechnology. TILT adenoviruses descibed above will be givenintratumorally to patients to enhance T-cell therapy (marked 4a, 5a).However, not all tumors can be reached through the intratumoral route.Thus, we have developed an Ad3 based delivery vehicle which can reachtumors through the intravenous route (marked as 4b, 5b).

FIG. 37 shows the structure of Ad3-hTERT-E3del-CMV-CD40L vector.Nucleotide sequence of the viral vector Ad3-hTERT-E3del-CMV-CD40L isshown in SEQ ID NO 30.

FIG. 38 shows the structure of Ad3-hTERT-E3del-E2F-CD40L vector.Nucleotide sequence of the viral vector Ad3-hTERT-E3del-E2F-CD40L isshown in SEQ ID NO 31.

FIG. 39 shows an agarose gel of pWEA-Ad3-hTERT-CMV-CD40L vector cut withrestriction enzymes. Correct restriction analyzes of the cloned virusvectors suggest correct DNA sequence for the virus.

FIG. 40 shows an agarose gel of pWEA-Ad3-hTERT-E2F-CD40L vector cut withrestriction enzymes. Correct restriction analyzes of the cloned virusvectors suggest correct DNA sequence for the virus. FIG. 41 showsfunctionality of E2F-CD40L and CMV-CD40L vectors in vitro. On thevertical axis the logarytmic scale of the relative visual titer theTCID₅₀ yields (PFU/ml). On the horizontal axis the days post infection(d). This showed that the viruses are functional and capable ofinfecting at least some tumour cell lines. The dilutions of virus werenot made according to the VP-titers. Progressive TCID₅₀: The newlyproduced viruses were first tested with progressive TCID₅₀ to determinewhether they have oncolytic properties. After nine (9) days ofincubation the infections became visible in all culture plates of A549cells, which indicated that all the new viruses were functional. Duringthe following days the infections continued spreading accordingly to theamount of virus pipetted per cell. Slight differences were detected inthe amount and speed of cell-lysis.

FIGS. 42-44 reveal that all oncolytic serotype 3 viruses showedsignificantly (P<0.05) better cell killing than the non-replicatingAd3eGFP control virus in A549 lung cancer cells, PC3-MM2 prostate cancercells and SKOV3 ovarian cancer cells. No significant difference betweenthe oncolytic Ad3 viruses could be seen suggesting that all virusconstructs are fully functional and that the E3 area deletion, theinserted promoters (CMV or E2F) or the inserted transgene (CD40L) do notaffect the oncolytic potency in vitro. FIG. 45 shows anti-tumor efficacyof Ad3 based viruses in vivo: orthotopic intraperitoneal ovarian cancermodel. Ad3-hTERT-E3del-E2F-CD40L had the best anti-tumor efficacy. ELISAconfirmed CD40L release into the blood stream.

FIG. 46 shows a therapeutic window of oncolytic adenovirus coding formurine CD40L in immunocompetent mice. DOSE 5: 1×10¹¹ VP/mouse; DOSE 4:3×10¹⁰ VP/mouse; DOSE 3: 1×10¹⁰ VP/mouse; DOSE 2: 1×10⁹ VP/mouse; DOSE1: 1×10⁸ VP/mouse; Positive control (DOSE 2 intratumorally.) With dose5, 67% of mice had signs of liver toxicity. Dose 4 was able to achievegood tumor transduction following i.v. delivery, without signs of livertoxicity.

FIG. 47 shows liver enzyme release in mice treated through theintravenous route with oncolytic adenovirus coding for murine CD40L inimmunocompetent mice. There was not much liver toxicity, as measured byliver enzyme release, in any intravenous treatment groups (DOSE 1-5).Last bar indicates DOSE 2 given intratumorally. However, in DOSE 5 therewas liver toxicity in visual inspection->DOSE 4 is maximum tolerateddose for intravenous delivery. (the doses from mock to dose 2 arerepresented as bars from left to right)

FIG. 48 shows mechanisms of enhancement of adoptive cell therapy by dualcytokine-expressing virus. Virus infection and innate sensing of virusparticles induces danger signals, which includes upregulation of HLA/MHCclass I molecules on cancer cells, activation and maturation of antigenpresenting cells and secretion of immune cell-recruiting cytokines.Danger signals are further amplified by tumor cell death with oncolyticviruses, which also releases tumor antigens and increases recognition oftumor tissue by the immune system. Viruses express two cytokines: the Tcell recruiting cytokine attracts adoptively transferred T cells intothe tumor, and the T cell expanding cytokine, in a specific embodimentinterleukin 2, increases and maintains their proliferation.

FIG. 49 shows schematics of the trafficking experiment with recombinantmouse cytokines. B16-OVA bearing C57BL/6 female mice are adoptivelytransferred with 2.0*10⁶ CD8a+ enriched OT-I lymphocytes (box) i.p. onday 0 and treated with intratumoral injections of recombinant murinecytokines (triangles) on workdays. Tumor growth is monitored andrecorded thrice a week (circles) by using electronic calipers. Mice aresacrificed (X) at two different time points (SAC1 and SAC2), tumors areharvested and samples are analyzed using OT-I qPCR and T-cell FACSanalysis.

FIG. 50 shows schematics of the trafficking experiment with adenovirusescoding for mouse cytokines. B16-OVA bearing C57BL/6 female mice areadoptively transferred with 2.0*10⁶ CD8a+enriched OT-I lymphocytes (box)i.p. on day 0 and intratumorally treated with adenoviruses armed withdifferent mouse cytokines (red triangles) on workdays. Tumor growth ismonitored and recorded thrice a week (circles) by using electroniccalipers. Mice are sacrificed (X) at two different time points (SAC1 andSAC2), tumors are harvested and samples analyzed using OT-I qPCR andT-cell FACS analysis.

FIG. 51 shows schematics of the trafficking experiment with using 111Inradiolabeled OT-I cells and SPECT/CT imaging. B16-OVA bearing C57BL/6female mice are intratumorally injected with 1e9 VP of 5/3 chimericvirus (triangles) on six consecutive days. First group of mice willreceive adoptive transfer of 2.0*10⁶ CD8a+enriched, indium oxine labeledOT-I lymphocytes (box) i.v. on day 0 and the other group of mice on day7. Accumulation of OT-I cells into tumors is quantitated by SPECT/CTimaging (circles). Mice are sacrificed (X) at two different time points(SAC1 and SAC2), tumors are harvested and their final radioactivity ismeasured.

DETAILED DESCRIPTION OF THE INVENTION Adoptive Cell Therapy

The general approach of the present invention is the development of atreatment for patients with cancer using the transfer of immunelymphocytes that are capable of reacting with and destroying the cancer.Isolated tumor infiltrating lymphocytes are grown in culture to largenumbers and infused into the patient. In the present inventionadenoviral vectors coding for at least one cytokine are utilized forincreasing the effect of lymphocytes. Separate administrations of anadoptive cell therapeutic composition and adenoviral vectors arefrequently preceded by myeloablating or non-myeloablatingpreconditioning chemotherapy and/or radiation. The adoptive cell therapytreatment is intended to reduce or eliminate cancer in the patient.(FIG. 21)

This invention relates to therapies with an adoptive cell therapeuticcomposition, e.g. tumor infiltrating lymphocytes, TCR modifiedlymphocytes or CAR modified lymphocytes. This invention relates toT-cell therapies in particular, but also other adoptive therapies suchas NK cell therapies or other cell therapies. Indeed, according to thepresent invention the adoptive cell therapeutic composition may compriseunmodified cells such as in TIL therapy or genetically modified cells.There are two common ways to achieve genetic targeting of T-cells totumor specific targets. One is transfer of a T-cell receptor with knownspecificity (TCR therapy) and with matched human leukocyte antigen (HLA,known as major histocompatibility complex in rodents) type. The other ismodification of cells with artificial molecules such as chimeric antigenreceptors (CAR). This approach is not dependent on HLA and is moreflexible with regard to targeting molecules. For example, single chainantibodies can be used and CARs can also incorporate co-stimulatorydomains. However, the targets of CAR cells need to be on the membrane oftarget cells, while TCR modifications can utilize intracellular targets.

As used herein “adoptive cell therapeutic composition” refers to anycomposition comprising cells suitable for adoptive cell transfer. In oneembodiment of the invention the adoptive cell therapeutic compositioncomprises a cell type selected from a group consisting of a tumorinfiltrating lymphocyte (TIL), TCR (i.e. heterologous T-cell receptor)modified lymphocytes and CAR (i.e. chimeric antigen receptor) modifiedlymphocytes. In another embodiment of the invention, the adoptive celltherapeutic composition comprises a cell type selected from a groupconsisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gammaT-cells, regulatory T-cells and peripheral blood mononuclear cells. Inanother embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells,delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclearcells form the adoptive cell therapeutic composition. In one specificembodiment of the invention the adoptive cell therapeutic compositioncomprises T cells. As used herein “tumor-infiltrating lymphocytes” orTILs refer to white blood cells that have left the bloodstream andmigrated into a tumor. Lymphocytes can be divided into three groupsincluding B cells, T cells and natural killer cells. In another specificembodiment of the invention the adoptive cell therapeutic compositioncomprises T-cells which have been modified with target-specific chimericantigen receptors or specifically selected T-cell receptors. As usedherein “T-cells” refers to CD3+ cells, including CD4+ helper cells, CD8+cytotoxic T-cells and γδ T cells.

In addition to suitable cells, adoptive cell therapeutic compositionused in the present invention may comprise any other agents such aspharmaceutically acceptable carriers, buffers, excipients, adjuvants,additives, antiseptics, filling, stabilising and/or thickening agents,and/or any components normally found in corresponding products.Selection of suitable ingredients and appropriate manufacturing methodsfor formulating the compositions belongs to general knowledge of a manskilled in the art.

The adoptive cell therapeutic composition may be in any form, such assolid, semisolid or liquid form, suitable for administration. Aformulation can be selected from a group consisting of, but not limitedto, solutions, emulsions, suspensions, tablets, pellets and capsules.The compositions are not limited to a certain formulation, instead thecomposition can be formulated into any known pharmaceutically acceptableformulation. The pharmaceutical compositions may be produced by anyconventional processes known in the art.

Viral Vectors

The oncolytic adenoviral vectors used in the present invention can beany adenoviral vectors suitable for treating a human or animal. In oneembodiment of the invention, the adenoviral vectors are vectors of humanviruses, and can be selected from a group consisting of

Ad5, Ad3 and Ad5/3 vectors. In another embodiment, the vector is Ad5 orAd5/3 vector.

As used herein “an oncolytic adenoviral vector” refers to an adenoviralvector capable of infecting and killing cancer cells by selectivereplication in tumor versus normal cells.

The vectors may be modified in any way known in the art, e.g. bydeleting, inserting, mutating or modifying any viral areas. The vectorsare made tumor specific with regard to replication. For example, theadenoviral vector may comprise modifications in E1, E3 and/or E4 such asinsertion of tumor specific promoters (eg. to drive E1), deletions ofareas (e.g. the constant region 2 of E1 as used in “D24”, E3/gp19k,E3/6.7k) and insertion of transgenes. Furthermore, fiber knob areas ofthe vector can be modified. In one embodiment of the invention theadenoviral vector is Ad5/3 comprising an Ad5 nucleic acid backbone andAd3 fiber knob or Ad5/3 chimeric fiber knob.

As used herein, expression “adenovirus serotype 5 (Ad5) nucleic acidbackbone” refers to the genome of Ad5. Similarly “adenovirus serotype 3(Ad3) nucleic acid backbone” refers to the genome of Ad3. “Ad5/3 vector”refers to a chimeric vector having parts of both Ad5 and Ad3 vectors. Ina specific embodiment of the invention, the capsid modification of thevector is Ad5/3 chimerism. As used herein, “Ad5/3 chimeric fiber knob”refers to a chimerism, wherein the knob part of the fiber is from Adserotype 3, and the rest of the fiber is from Ad serotype 5.Specifically, in one embodiment, the construct has the fiber knob fromAd3 while the remainder of the genome is from Ad5. (See FIGS. 17, 33 and34)

One approach for generation of a tumor specific oncolytic adenovirus isengineering a 24 base pair deletion (D24) affecting the constant region2 (CR2) of E1. In wild type adenovirus CR2 is responsible for bindingthe cellular Rb tumor suppressor/cell cycle regulator protein forinduction of the synthesis (S) phase i.e. DNA synthesis or replicationphase. The interaction between pRb and E1A requires eight amino acids121 to 127 of the E1A protein conserved region, which are deleted in thepresent invention. The vector of the present invention comprises adeletion of nucleotides corresponding to amino acids 122-129 of thevector according to Heise C. et al. (2000, Nature Med 6, 1134-1139).Viruses with the D24 are known to have a reduced ability to overcome theG1-S checkpoint and replicate efficiently only in cells where thisinteraction is not necessary, e.g. in tumor cells defective in theRb-p16 pathway, which includes most if not all human tumors. (See FIGS.17, 33 and 34)

It is also possible to replace E1A endogenous viral promoter for exampleby a tumor specific promoter. In a specific embodiment of the inventionhTERT promoter is utilized in the place of E1A endogenous viralpromoter.

The E3 region is nonessential for viral replication in vitro, but the E3proteins have an important role in the regulation of host immuneresponse i.e. in the inhibition of both innate and specific immuneresponses. The gp19k/6.7K deletion in E3 refers to a deletion of 965base pairs from the adenoviral E3A region. In a resulting adenoviralconstruct, both gp19k and 6.7K genes are deleted (Kanerva A et al. 2005,Gene Therapy 12, 87-94). The gp19k gene product is known to bind andsequester major histocompatibility complex I (MHC1, known as HLA1 inhumans) molecules in the endoplasmic reticulum, and to prevent therecognition of infected cells by cytotoxic T-lymphocytes. Since manytumors are deficient in HLA1/MHC1, deletion of gp19k increases tumorselectivity of viruses (virus is cleared faster than wild type virusfrom normal cells but there is no difference in tumor cells). 6.7Kproteins are expressed on cellular surfaces and they take part in downregulating TNF-related apoptosis inducing ligand (TRAIL) receptor 2.(See FIGS. 17, 33 and 34)

Both of these deletions provide a surprising advantage with regard toour invention. Since we are attempting to regain expression of HLA/MHCfor presentation of tumor epitopes to the adoptively transferredT-cells, expression of the gp19k protein is counterproductive and infact the up regulation of HLA/MHC requires deletion of gp19k. Withregard to 6.7k, since an embodiment of our invention is production ofTNFalpha from the virus, and one of its anti-tumor activities is adirect anti-tumor proapoptotic effect (on both transduced andnon-transduced bystander cells), the presence of 6.7k iscounterproductive.

In one embodiment of the invention, the cytokine transgene or transgenesare placed into a gp19k/6.7k deleted E3 region, under the E3 promoter.This restricts transgene expression to tumor cells that allowreplication of the virus and subsequent activation of the E3 promoter.E3 promoter may be any exogenous (e.g. CMV or E2F promoter) orendogenous promoter known in the art, specifically the endogenous E3promoter. Although the E3 promoter is chiefly activated by replication,some expression occurs when E1 is expressed. As the selectivity of D24type viruses occurs post E1 expression (when E1 is unable to bind Rb),these viruses do express E1 also in transduced normal cells. Thus, it isof critical importance to regulate also E1 expression to restrict E3promoter mediated transgene expression to tumor cells.

In another embodiment of the invention E3 gp19k/6.7k is kept in thevector but one or many other E3 areas have been deleted (e.g. E3 9-kDa,E3 10.2 kDa, E3 15.2 kDa and/or E3 15.3 kDa).

In a specific embodiment of the invention the oncolytic adenoviralvector is based on an adenovirus serotype 5 (Ad5) nucleic acid backbonecomprising a 5/3 chimeric fiber knob, and comprising the following: E2F1promoter for tumor specific expression of E1A, a 24 bp deletion (D24) inthe Rb binding constant region 2 of adenoviral E1, a nucleic acidsequence deletion of viral gp19k and 6.7k reading frames, with atransgene insertion into the deleted region, resulting inreplication-associated control of transgene expression under the viralE3 promoter, and a nucleic acid sequence encoding at least one cytokinetransgene in the place of the deleted adenoviral genes gp19k/6.7K in theE3 region (FIG. 17). In one embodiment of the invention, the adenoviralvector is based on a human adenovirus. (See FIGS. 17, 33 and 34)

In another specific embodiment of the invention the oncolytic adenoviralvector is based on an adenovirus serotype 3 (Ad3) nucleic acid backbone,and comprises the following: a deletion in the E3 area, and a tumorspecific promoter (e.g. CMV or E2F) for expression of a transgene (e.g.CD40L) in the place of the deleted area of E3. In one embodiment of theinvention, the adenoviral vector is based on a human adenovirus. (SeeFIGS. 37 and 38, corresponding nucleotide sequences of the viral vectorsAd3-hTERT-E3del-CMV-CD40L and Ad3-hTERT-E3del-E2F-CD40L is shown in SEQID NOs 30 and 31)

The exact functions of the Early Region (E3) proteins in adenovirus 3are not known. Generally in adenoviruses they do not seem to impairreplication when deleted and they seem to affect anti-viral hostresponse to adenoviruses (Wold et al., 1999). The E3 of the humanadenovirus genome contains the highest level of genetic diversity amongthe six species (A-F) of adenoviruses found in humans. This diversity ingenetic content is primarily located between the highly conservedE3-gp19K and E3-RIDα open reading frames (ORFs) where species-specificarrays of genes are encoded (Burgert and Blusch, 2000).

Cytotoxic T-cell mediated killing of viral-infected cells is modulatedby E3-gp19K. This is accomplished by blocking transport of MHC class Ito the plasma membrane, and inhibiting the TAP-MHC class I complexformation (Andersson et al., 1985; Andersson et al., 1987; Burgert andKvist, 2002, Bennet et al., 1999).

Thus, in one aspect of the invention the important molecule E3-gp19K iscomprised in the adenoviral vector to make virus replication morestealty and enable more time for oncolysis and its beneficial effects.Also, retaining E3-gp19K can reduce induction ofanti-adenovirus-cytotoxic T-cells, resulting in more anti-tumor T-cells.

In one embodiment of the invention the oncolytic adenoviral vector isbased on an adenovirus serotype 3 (Ad3) nucleic acid backbone, andcomprises the following: a promoter (e.g. hTERT) for tumor specificexpression of E1A, a deletion in the E3 area (e.g. a deletion affectingE3 9-kDa, E3 10.2 kDa, E3 15.2 kDa and E3 15.3 kDa), and a tumorspecific promoter (e.g. CMV or E2F) for expression of a transgene (e.g.CD40L) in the place of the deleted area of E3. In one embodiment of theinvention, the nucleic acid backbone of the vector is fully adenovirusserotype 3. In one embodiment of the invention in Ad3 delE3 viruses thefollowing features have been deleted: E3 9-kDa, E3 10.2-kDa, E315.2-kDa, E3 15.3-kDa and furthermore, CD40L with a promoter (CMV orE2F) has been inserted in their place. These viruses induce apoptosis oftumor cells and triggers several immune mechanisms, including a T-helpertype 1 (TH1) response, which leads to activation of cytotoxic T cellsand reduction of immunosuppression.

Cytokines participate in immune response by acting through variousmechanisms including recruitment of T-cells towards the tumor. Thenucleotide sequence encoding a cytokine transgene may be from any animalsuch as a human, ape, rat, mouse, hamster, dog or cat, but specificallyit is encoded by a human sequence. The nucleotide sequence encoding thetransgene may be modified in order to improve its effects, or unmodifiedi.e. of a wild type.

Particular embodiments of the present invention include viral vectorscoding for at least one cytokine. Cytokines used in the presentinvention can be selected from any known cytokines in the art. In oneembodiment of the invention the cytokine is selected from a groupconsisting of interferon alpha, interferon beta, interferon gamma,complement C5a, IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1,CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16,CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2,CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4,CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7,CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12,CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6,CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 andXCL2. In a specific embodiment of the invention the cytokine is IL-2 orTNFalpha. In another embodiment of the invention the cytokine orcytokines is/are selected from a chemokine group consisting of CCL1,CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16,CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2,CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4,CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7,CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12,CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6,CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 andXCL2.

The viral vectors of the invention may code for one, two, three, four,five or more cytokines. In one embodiment of the invention the oncolyticadenoviral vector codes for two or more cytokines, most specificallytwo. These two cytokines may be any known cytokines, for exampleincluding but not limited to the ones listed above, with the addition ofGMCSF. The two cytokines may be different cytokines. In one embodimentof the invention the oncolytic adenoviral vector codes for any two ormore cytokines selected from a cytokine group consisting of interferonalpha, interferon beta, interferon gamma, complement C5a, GMCSF, IL-2,TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13,CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19,CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1,CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6,CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2,CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15,CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9,CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2, or the oncolyticadenoviral vector codes for IL-2 and a cytokine or cytokines selectedfrom a cytokine group consisting of interferon alpha, interferon beta,interferon gamma, complement C5a, GMCSF, TNFalpha, CD40L, IL12, IL-23,IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3,CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28,CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2,CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4,CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5,CXCR6, CXCR7 and XCL2. In a specific embodiment of the invention thecytokines are IL-2 and TNFalpha. The other cytokine functions byattracting and activating the T cells and reducing tumorimmunosuppression, while IL-2 induces the propagation of the T-cellgraft. Thus, IL-2 is produced locally at the tumor where it is needed,instead of injected systemically as is typically done in T-cell therapy,which can cause side effects, and therefore a major problem of the priorart therapies (i.e. toxicity of systemic IL-2) can be prevented by thisembodiment.

The danger signaling provided by replication of the oncolytic virus, andactivation of pathogen associated molecular pattern recognitionreceptors by viral DNA, together with the action of the transgene(s) mayreduce tumor immunosuppression to such degree that preconditioningtherapy can be omitted. Consequently, and major issue in prior art,toxicity due to preconditioning chemotherapy and radiation can beavoided.

In one embodiment of the invention the virus vector comprises aninternal ribosomal entry site (IRES) or optionally a ribosome shunt site2A between the two transgenes. Thus, IRES or a ribosome shunt site 2Amay be between any cytokines, such as IL-2 and any other cytokineselected from the above listed cytokine group. As used herein “IRES”refers to a nucleotide sequence that enables initiation of thetranslation in the middle of a messenger RNA sequence in proteinsynthesis. IRES can be from any virus, but in one embodiment of theinvention IRES is from encephalomyocarditis virus (EMCV). As used herein“a ribosome shunt site 2A” refers to a translation initiation site inwhich ribosomes physically bypass parts of the 5′ untranslated region toreach the initiation codon. Both the IRES and the A2 enable viruses toproduce two transgenes from one promoter (the E3 promoter).

Schematics of the general layouts of the virus genomes, which may beused in the present invention, are shown in FIGS. 17, 33, 34, 37 and 38.Nucleotide sequences of the viral vectors comprising transgenes C5a,hCD40L, hIFNa2, hIFNb1, hIFNg1, hIL2 or TNFalpha are shown in SEQ ID NOs1-7, respectively (Ad5/3-E2F-D24-transgene). Nucleotide sequences of theviral vectors comprising CD40L are also shown in SEQ ID NOs 30 and 31(Ad3-hTERT-E3del-CMV-CD40L and Ad3-hTERT-E3del-E2F-CD40L). Furthermore,nucleotide sequences of the viral vectors comprising two transgenes, theother one being IL-2 and the other one C5a, CD40L, IFNa2, IFNb, IFNg,GMCSF or TNFalpha, are shown in SEQ ID NOs 8-21 (SEQ ID NO: 8C5a-2A-IL2, SEQ ID NO: 9 IFNa-2A-IL2, SEQ ID NO: 10 TNFalpha-2A-IL2, SEQID NO: 11 CD40L-2A-IL2, SEQ ID NO: 12 IFNb-2A-IL2, SEQ ID NO: 13GMCSF-2A-IL2, SEQ ID NO: 14 IFNg-2A-IL2, SEQ ID NO: 15 C5a-IRES-IL2, SEQID NO: 16 IFNa-IRES-IL2, SEQ ID NO: 17 TNFalpha-IRES-IL2, SEQ ID NO: 18CD40L-IRES-IL2, SEQ ID NO: 19 IFNb-IRES-IL2, SEQ ID NO: 20GMCSF-IRES-IL2, SEQ ID NO: 21 IFNg-IRES-IL2)(Ad5/3-E2F-D24-transgene-IRES/2A-transgene).

In summary, the key advantages of the present invention utilizing viralvectors comprising at least one cytokine transgene are: i) cytokines andvirus per se cause a danger signal which recruits T cells and otherimmune cells to tumors, ii) cytokines induce T cell proliferation bothat the tumor and in local lymphoid organs, iii) cytokines and virus perse are able to induce T cells (both the adoptive T-cell graft andnatural, innate anti-tumor T-cells) to propagate at the tumor, iv)cytokine and/or virus induce the up regulation of antigen-presentingmolecules (HLA) on cancer cells, rendering them sensitive to recognitionand killing by T cells, and v) cytokines and virus replication favorablyalter tumor microenvironment by reducing immunosuppression and cellularanergy.

The viral vectors utilized in the present inventions may also compriseother modifications than described above. Any additional components ormodifications may optionally be used but are not obligatory for thepresent invention.

Insertion of exogenous elements may enhance effects of vectors in targetcells. The use of exogenous tissue or tumor-specific promoters is commonin recombinant vectors and they can also be utilized in the presentinvention.

In summary, the present invention reveals that the replication ofoncolytic virus can recruit T-cells and induce danger signals at thetumor, reducing immunosuppression and cellular anergy. These effects aremediated through pathogen associated molecular pattern recognitionreceptors, an evolutionarily conserved mechanism for inducing immunityand not subject to tolerance. The present invention also reveals that anadded benefit of the oncolytic platform, capable of replication intumors but not normal cells, is self-amplification at the tumor. Inaddition, the oncolytic effect per se may add to the overall anti-tumoreffect in humans.

Cancer

The recombinant vectors of the present invention are replicationcompetent in tumor cells. In one embodiment of the invention the vectorsare replication competent in cells, which have defects in theRb-pathway, specifically Rb-p16 pathway. These defective cells includeall tumor cells in animals and humans. As used herein “defects in theRb-pathway” refers to mutations and/or epigenetic changes in any genesor proteins of the pathway. Due to these defects, tumor cellsoverexpress E2F and thus, binding of Rb by E1A CR2, that is normallyneeded for effective replication, is unnecessary. Further selectivity ismediated by the E2F promoter, which only activates in the presence offree E2F, as seen in Rb/p16 pathway defective cells. In the absence offree E2F, no transcription of E1A occurs and the virus does notreplicate. Inclusion of the E2F promoter is important to preventexpression of E1A in normal tissues, which can cause toxicity bothdirectly and indirectly through allowing transgene expression from theE3 promoter.

The present invention relates to approaches for treating cancer in asubject. In one embodiment of the invention, the subject is a human oran animal, specifically an animal or human patient, more specifically ahuman or an animal suffering from cancer.

The approach can be used to treat any cancers or tumors, including bothmalignant and benign tumors, both primary tumors and metastases may betargets of the approach. In one embodiment of the invention the cancerfeatures tumor infiltrating lymphocytes. The tools of the presentinvention are particularly appealing for treatment of metastatic solidtumors featuring tumor infiltrating lymphocytes. In another embodimentthe T-cell graft has been modified by a tumor or tissue specific T-cellreceptor of chimeric antigen receptor.

As used herein, the term “treatment” or “treating” refers toadministration of at least oncolytic adenoviral vectors or at leastoncolytic adenoviral vectors and adoptive cell therapeutic compositionto a subject, preferably a mammal or human subject, for purposes whichinclude not only complete cure but also prophylaxis, amelioration, oralleviation of disorders or symptoms related to a cancer or tumor.Therapeutic effect may be assessed by monitoring the symptoms of apatient, tumor markers in blood or for example a size of a tumor or thelength of survival of the patient

In another embodiment of the invention the cancer is selected from agroup consisting of nasopharyngeal cancer, synovial cancer,hepatocellular cancer, renal cancer, cancer of connective tissues,melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer,colorectal cancer, brain cancer, throat cancer, oral cancer, livercancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma,pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, neuroma, vonHippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, analcancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer,oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bonecancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer ofunknown primary site, carcinoid, carcinoid of gastrointestinal tract,fibrosarcoma, breast cancer, Paget's disease, cervical cancer,colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer,head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, livercancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicularcancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skincancer, mesothelioma, multiple myeloma, ovarian cancer, endocrinepancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer,penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma,small intestine cancer, stomach cancer, thymus cancer, thyroid cancer,trophoblastic cancer, hydatidiform mole, uterine cancer, endometrialcancer, vagina cancer, vulva cancer, acoustic neuroma, mycosisfungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer,heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer,palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer,pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

Before classifying a human or animal patient as suitable for the therapyof the present invention, the clinician may examine a patient. Based onthe results deviating from the normal and revealing a tumor or cancer,the clinician may suggest treatment of the present invention for apatient.

Pharmaceutical Composition

A pharmaceutical composition of the invention comprises at least onetype of viral vectors of the invention. Furthermore, the composition maycomprise at least two, three or four different vectors. In addition tothe vector, a pharmaceutical composition may also comprise othertherapeutically effective agents, any other agents such aspharmaceutically acceptable carriers, buffers, excipients, adjuvants,additives, antiseptics, filling, stabilising and/or thickening agents,and/or any components normally found in corresponding products.Selection of suitable ingredients and appropriate manufacturing methodsfor formulating the compositions belongs to general knowledge of a manskilled in the art.

The pharmaceutical composition may be in any form, such as solid,semisolid or liquid form, suitable for administration. A formulation canbe selected from a group consisting of, but not limited to, solutions,emulsions, suspensions, tablets, pellets and capsules. The compositionsof the current invention are not limited to a certain formulation,instead the composition can be formulated into any knownpharmaceutically acceptable formulation. The pharmaceutical compositionsmay be produced by any conventional processes known in the art.

In one embodiment of the invention, the viral vector or pharmaceuticalcomposition acts as an in situ vehicle for recruitment of T-cells,enhancing their therapeutic effect and allowing their propagation at thetumor.

A pharmaceutical kit of the present invention comprises an adoptive celltherapeutic composition and oncolytic adenoviral vectors coding for atleast one cytokine. The adoptive cell therapeutic composition isformulated in a first formulation and the oncolytic adenoviral vectorscoding for at least one cytokine are formulated in a second formulation.In another embodiment of the invention the first and the secondformulations are for simultaneous or sequential, in any order,administration to a subject

Administration

The vector or pharmaceutical composition of the invention may beadministered to any eukaryotic subject selected from a group consistingof plants, animals and human beings. In a specific embodiment of theinvention, the subject is a human or an animal. An animal may beselected from a group consisting of pets, domestic animals andproduction animals.

Any conventional method may be used for administration of the vector orcomposition to a subject. The route of administration depends on theformulation or form of the composition, the disease, location of tumors,the patient, comorbidities and other factors.

In one embodiment of the invention the separate administration(s) ofadoptive cell therapeutic composition and oncolytic adenoviral vectorscoding for at least one cytokine to a subject is (are) conductedsimultaneously or consecutively, in any order. As used herein “separateadministration” or “separate” refers to a situation, wherein adoptivecell therapeutic composition and oncolytic adenoviral vectors are twodifferent products or compositions distinct from each other.

Only one administration of adoptive cell therapeutic composition andoncolytic adenoviral vectors coding for at least one cytokine of theinvention or only oncolytic or non-cytolytic virus vectors may havetherapeutic effects. There may be any period between the administrationsdepending for example on the patient and type, degree or location ofcancer. In one embodiment of the invention there is a time period of oneminute to four weeks, specifically 1 to 10 days, more specifically 1 tofive days, between the consecutive administration of adoptive celltherapeutic composition and oncolytic adenoviral vectors coding for atleast one cytokine and/or there are several administrations of adoptivecell therapeutic composition and oncolytic adenoviral vectors. Thenumbers of administration times of adoptive cell therapeutic compositionand oncolytic adenoviral vectors may also be different during thetreatment period. Oncolytic adenoviral vectors or pharmaceutical oradoptive cell compositions may be administered for example from 1 to 10times in the first 2 weeks, 4 weeks, monthly or during the treatmentperiod. In one embodiment of the invention, administration of vectors orany compositions is done three to seven times in the first 2 weeks, thenat 4 weeks and then monthly. In a specific embodiment of the invention,administration is done four times in the first 2 weeks, then at 4 weeksand then monthly. The length of the treatment period may vary, and forexample may last from two to 12 months or more.

In a specific embodiment of the invention an adoptive cell therapeuticcomposition and oncolytic adenoviral vectors are administered on thesame day and thereafter oncolytic adenoviral vectors are administeredevery week, two weeks, three weeks or every month during a treatmentperiod which may last for example from one to 6 or 12 months or more.

In one embodiment of the invention, the administration of oncolyticvirus is conducted through an intratumoral, intra-arterial, intravenous,intrapleural, intravesicular, intracavitary or peritoneal injection, oran oral administration. Any combination of administrations is alsopossible. The approach can give systemic efficacy despite localinjection. Adoptive cell therapeutic composition may be administeredintravenously or intratumorally. In one embodiment the administration ofthe adoptive cell therapeutic composition and/or oncolytic viral vectorscoding for at least one cytokine is conducted through an intratumoral,intra-arterial, intravenous, intrapleural, intravesicular, intracavitaryor peritoneal injection, or an oral administration. In a specificembodiment of the invention TILs or T cells are administeredintravenously and viral vectors intratumorally and/or intravenously. Ofnote, virus is delivered to the tumor separately from administration ofT-cells; virus is not used to modify the T-cell graft ex vivo. Inessence, the virus modifies the tumor in such a way that the T-cellgraft can work better.

The effective dose of vectors depends on at least the subject in need ofthe treatment, tumor type, location of the tumor and stage of the tumor.The dose may vary for example from about 1×10⁸ viral particles (VP) toabout 1×10¹⁴ VP, specifically from about 5×10⁹ VP to about 1×10¹³ VP andmore specifically from about 8×10⁹ VP to about 1×10¹² VP. In oneembodiment oncolytic adenoviral vectors coding for at least one cytokineare administered in an amount of 1×10¹⁰-1×10¹⁴ virus particles. Inanother embodiment of the invention the dose is in the range of about5×10¹⁰-5×10¹¹ VP.

The amount of cells transferred will also depend on the patient, buttypical amounts range from 1×10⁹-1×10¹² cells per injection. The numberof injections also varies but typical embodiments include 1 or 2 roundsof treatment several (e.g. 2-4) weeks apart.

Any other treatment or combination of treatments may be used in additionto the therapies of the present invention. In a specific embodiment themethod or use of the invention further comprises administration ofconcurrent or sequential radiotherapy, monoclonal antibodies,chemotherapy or other anti-cancer drugs or interventions (includingsurgery) to a subject.

The terms “treat” or “increase”, as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orincrease. Rather, there are varying degrees of which one of ordinaryskill in the art recognizes as having a potential benefit or therapeuticeffect. In this respect, the present inventive methods can provide anyamount of increase in the efficacy of T-cell therapy or any degree oftreatment or prevention of a disease.

FIGS. 28-32, 36 and 48 illustrate the methods and mechanisms of thepresent invention.

It will be obvious to a person skilled in the art that, as thetechnology advances, the inventive concept can be implemented in variousways. The invention and its embodiments are not limited to the examplesdescribed above but may vary within the scope of the claims.

EXAMPLES Materials & Methods

B16-OVA animal model: ovalbumin-expressing B16 cells (B16-OVA) weremaintained in RPMI, 10% FBS, 5 mg/ml G418, 20 mM L-Glutamine, 1×Pen/Strep solution (GIBCO). 4-7-week-old C57BL/6 immunocompetent femalemice were implanted subcutaneously with 2.5×10⁵ B16-OVA cells in 50 ulRPMI, 0% FBS, in the right flank, one tumor per mouse. Roughly ten dayspost tumor implantation (when tumors became injectable, ˜3 mm minimumdiameter), mice were divided into groups and treated in some experimentson six consecutive days with intratumoral injections of either 50 ul PBSor 1×10⁹viral particles (VPs) of oncolytic adenovirus in 50 ul PBS. Inother experiments, three injections were given on days 0, 2 and 4. Asmurine cells are non-permissive to human adenovirus, multipleintratumoral virus injections were used to mimic virusreplication-induced inflammation, (Blair et al., 1989).

Adoptive transfer: On the first day of the i.t. treatment, the mice alsoreceived by adoptive transfer in the intraperitoneal cavity 5×10⁵ to2×10⁶ overnight-rested CD8a-enriched and expanded splenocytes from4-8-week-old C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) mice, geneticallyengineered to have only ovalbumin (OVA)-specific CD8 T-cell receptors,in 100 ul RPMI, 0% FBS. CD8a-enrichment was performed by mouse CD8a(Ly-2) MicroBeads 5 days prior to transfer, per manufacturer'sinstructions (Miltenyi Biotech, USA, cat. no 130-049-401). Enrichedcells were expanded in numbers for five days in lymphocyte medium (RPMI,10% FBS, 20 mM L-Glutamine, 1× Pen/Strep solution, 15 mM HEPES, 50 μM2-mercaptoethanol, 1 mM Na pyruvate) in the presence of recombinantmurine IL-2 (160 ng/ml) and soluble anti-mouse CD3E antibody (0.3 ug/ml,Abcam, clone 145-2C11).

Tissue processing for flow cytometry: Mice were euthanized and spleens,draining lymph nodes and tumors were harvested in 1 to 10 ml RPMI, 10%FBS, and blood was collected by terminal heart bleed into the pleuralcavity and transferred by disposable syringe into EDTA-containingmicrocentrifuge tubes, and processed for analysis: solid tissues wereroughly dissociated by scalpel and triturated in a 10 ml disposablesterile pipette tip in 5 to 10 ml ACK lysing buffer (150 mM NH₄C1, 10 mMKHCO₃, 0.1 mM EDTA, pH 7.2) and incubated at room temperature (RT) for˜20 minutes, upon which cells were pelleted at 1200 rpm 5 min+4° C.,following which cells were re-suspended in 1 to 10 ml RPMI, 10% FBS,depending on the estimated amount of cells, and passed through a 40 μmsterile filter to create a single-cell solution. In some experiments,tumor tissue was instead processed directly after scalpel cutting(before addition of ACK) in 1 ml total volume of protease-coctail (RPMIsupplemented with collagenase type A, H or P, Roche, at 1 mg/ml andbenzonase, 125 units/ml final conc., Sigma, E1014-25KU) for 1-2 hours at37° C., 5% CO₂, after which 10 ml ACK lysing buffer was added and cellswere treated as above. 200 μl whole blood was pipetted into 5 ml ACKlysing buffer and treated as above. Cells were either incubatedovernight at 37° C., 5% CO₂, or analyzed directly by immunostaining andflow cytometry.

Tissue processing for cytokine analysis: Mice were euthanized and ˜2-10mm³ tumor pieces were frozen in 2 ml microcentrifuge tubes on dry iceand stored at −80° C. Tumor pieces were weighed and 200 μl ice-cold PBSadded. Pieces were homogenized by Tissue Master 125 rotor, 1× proteaseinhibitor cocktail (Sigma) and 0.1% BSA final conc. was added and tubeswere kept on ice. Tumor homogenate was spun at 2000 rpm 10 min+4° C. andthe supernatant was analyzed with CBA Flex Set cytokine beads (BD, USA)on BD FACSArray, per manufacturer's instructions.

Experiments Supporting the Invention Experiment 1 (Cytokines andChemokines Induced by Intratumoral Adenovirus Injection)

To study whether adenovirus infection could result in cytokine andchemokine expression, we injected mice harboring subcutaneous B16-OVAtumors intratumorally with either PBS or 5/3 chimeric oncolyticadenovirus on days 0, 1, 2, 3, 4 and 5. Tumors from three mice pertreatment group were extracted and processed for cytokine analysis onday 0 (before virus injection =baseline control), and from three othermice per time point on days 6, 10, 14 and 18.

Remarkably, the results showed a virus-induced increase in secretion ofIFN-γ and subsequent up-regulation of IFN-γ inducible chemokines RANTES,MIP-1a and MCP-1 on day 10 (FIG. 1).

For enhancing therapeutic efficacy of adoptive cell therapy, thesefindings are important

Based on this data, treatment with oncolytic cytokine-armed adenovirusresults in favorable alteration of tumor microenvironment, increasedchemotaxis of adoptively transferred immune cells and enhanced tumorcell recognition by cytotoxic CD8+ T-cells.

Experiment 2 (Adenovirus-Mediated Enhancement of Adoptive T CellTherapy)

To study the impact of adenovirus treatment on adoptive T cell therapy,murine B16-OVA melanoma tumors were treated with 5/3 chimeric oncolyticadenovirus alone or in combination with adoptive transfer oftumor-specific OT-I cells and compared to mice receiving intratumoralPBS injections. The results of three independent experiments summarizedin FIG. 2 reveal, on one hand, that virus injections on their own(keeping in mind that human adenovirus does not productively replicatein mouse cells) resulted in minor tumor growth control, lasting untilday 14 post-treatment and diminishing after that (FIG. 2A). On the otherhand, when treated mice were adoptively transferred with 5×10⁵ or 2×10⁶OT-I cells, statistically significant differences between PBS and Adgroups were obtained in two separate experiments (FIG. 2B and 2C,respectively).

Thus, the presence of virus in the tumor had a strong enhancing effecton adoptive cell therapy. Six intratumoral virus injections at 1×10⁹ VPseach in our hands gave in combination with adoptive transfer of OT-Icells equal or superior anti-tumor efficacy compared to what wasreported by Song et al. (2011, Mol Ther) for a single intramuscularinjection of 1×10¹⁰ VP of OVA-expressing replication-defectiveadenovirus (Ad-OVA) admixed with an equal amount of adenovirusco-expressing an A20-specific short-hairpin RNA and a secretory form offlagellin that stimulates toll-like receptor 5 (Ad-shAF) in the B16.OVAmelanoma model (Song XT et al. Mol Ther. 2011 January; 19(1):211-7,PMID: 20959814). In light of these results, a novel aspect of ourinvention is to target the virus injection into the tumor, where we canachieve even with unarmed virus superior tumor control tomulti-immune-functional armed virus administered intramuscularly.

Experiment 3 (Adenovirus-Mediated Alterations in Quality and Quantity ofImmune Cell Populations In Vivo)

To study the trafficking and proliferation of adoptively transferredcells of experiment 2, OT-I cells were stained ex vivo with 5 μMcarboxyfluorescein succinimidyl ester (CFSE). This fluorescent cellstaining dye is diluted with every cell division and therefore enablesus to trace lymphocyte proliferation by flow cytometry by analyzing ˜½fractional reduction of fluorescence signal intensity at each celldivision (up to 7 divisions, here labeled MO-7). On day 1 post-transferthe results showed virus-induced accumulation of transferred OT-I cells(CD8+ CFSE+double positive population) in the tumors, concomitant withreduction of these cells in the blood (FIG. 3A). At later time points,also the total CD8+ T-cell count appeared higher in the virus-treatedtumors compared to PBS-injected tumors, and on day 14 the overall CD8+T-cell count was increased in lymphoid organs of virus treated mice.

The amounts of OT-I cell divisions at different time points are depictedin FIG. 3B. Since the proliferation status of OT-I cells was the samebetween both groups on day 1, differences in the CD8+ cell count invarious organs were due to adenovirus-induced immune cell trafficking.At later time points, however, the situation had changed and theincrease of OT-I cells in the adenovirus treated tumor was due toincreased lymphocyte proliferation. On day 6 the majority of OT-I cellsin PBS treated tumors were arrested in M5 phase, whereas transferredcells in adenovirus group continued to proliferate (divisions M6-M7).This data suggests that oncolytic virotherapy or non-cytolytic virusinfection results in enhanced trafficking and proliferation ofadoptively transferred lymphocytes through breaking the immunesuppression in the tumors, attracting immune cells that contribute toCD8+ cell activation and/or through some other important mechanisms thathelp overcome T-cell anergy.

As support to our findings in animal models, we have observed transientdepression of blood lymphocyte counts during the first day followingoncolytic virus administration to patients with advanced cancer (FIG.4), suggesting mobilization of circulating T cells in response to acuteadenovirus infection in the tumor.

Furthermore, in support of adenovirus infection recruiting T cells intotumors, we detected increased numbers of CD8+ T cells in tumor biopsytissue sections after treatment than before (FIG. 5).

FIG. 6 shows results of adenovirus injections combined with adoptivetransfer of T cells.

FIG. 7 reveals dramatic increase in the number of “natural” anti-tumorT-cells due to adoptive transfer and virus injection.

FIG. 8 shows activated CD8+ cells in tumor and TIM-3 expression in thetumor on day 14.

FIG. 9 shows that increase in anti-tumor T-cells and reduction ofimmunosuppression results in systemic immunity against tumor antigens.

FIG. 10 shows distribution of OTI T-cells following virus injection.

FIG. 11 reveals that lifting immunosuppression can induce propagation ofcells.

FIG. 12 shows efficacy of recombinant cytokines (no virus) incombination with OT1 cells.

Experiment 4 (Adoptively Transferred T-Cells+Murine Cytokine-Armed Ad5Adenovirus)

Model:

C57BL/6 with B16-OVA (0.25×10e6 cells per animal)

Groups:

No injection

Ad5-Luc

Ad5-CMV-mTNFa

Ad5-CMV-mIFNg

Ad5-CMV-mIL2

Ad5-CMV-mIFNb1

No injection+OT1

Ad5-Luc+OT1

Ad5-CMV-mTNFa+OT1

Ad5-CMV-mIFNg+OT1

Ad5-CMV-mIL2+OT1

Ad5-CMV-mIFNb1+OT1

Ad5 vector is a vector of non-replicative human adenovirus coding for amouse transgene. The constructs were made with AdEasy technology(Agilent Inc); the transgene cassette (driven by a CMV promoter) is inthe deleted E1 region (see e.g. Diaconu I et al. Cancer Res. 2012 May 1;72(9):2327-38).

Group size:

n=7, 12×7=84 (+extra 20%=100)

Treatment schedule:

OT1 cells: 2×10e6 per animal i.p. on Day 1

Virus injections: 1×10e9 virus particles (OD260) on Day 1 and weeklythereafter Endpoint:

Tumor volume (measured every 2 days for the first week and then every 3days)

Collection of tumors and spleens when mice die or are killed; for FACSand/or ELISPOT (focus on assays most relevant according to Siri data).

The best transgenes in combination with T-cell therapy were TNFalpha jaIL2 (FIG. 13). Strengthening the data, the same cytokines wereimplicated in the experiment without virus.

FIG. 14 shows the results of different viruses (without T-cell therapy)on tumor size (FIG. 14).

FIG. 15 shows the excellent results of T-cell therapy in combinationwith Ad5-CMV-mTNFalpha-vector.

FIG. 16 shows the excellent results of T-cell therapy in combinationwith Ad5-CMV-mIL2-vector.

Novel Virus Constructs

The following new virus constructs are presented as examples of ourproposed technology:

C5a and TNF-α Expressing Oncolytic Viruses

We generated new oncolytic Ad5/3 adenoviruses carrying the activeportion of complement component C5a or human TNF-α as transgenes insteadof 6.7K/gp19 gene regions (FIG. 17).

Experiment 5 (Transgene Expression from C5a-Encoding Adenovirus Vector)

In order to confirm—as proof-of-concept—that oncolytic adenoviruses areable to express the chosen cytokines proposed to augment adoptive celltherapy, we infected human A549 cells in culture at 10 VP/cell ofadenovirus encoding C5a (FIG. 24), and assessed C5a levels in cellculture supernatant at different time points post infection by ELISA.Results indeed validate the assumption and support the generation ofproprietary adenovirus constructs harboring selected cytokines.

Experiment 6 (Effect on Monocyte Migration by Novel Adenovirus Vectors)

We tested the C5a capability of recruiting monocytes using an in vitrochemotaxis assay: A549 cells were infected either with adenovirusexpressing C5a or with unarmed control virus (10 VP/cell—infectiousunits between viruses similar), or were treated with PBS, and media wascollected 48 h post infection and was used to recruit human monocyticcell line THP1 in a transwell chemotaxis assay per manufacturer'sinstructions (Millipore QCM, cat. no.

ECM512). Results reveal significantly greater attraction of monocytes bysupernatant from C5a-expressing virus-infected cells than by medium fromnon-infected cells or cells infected with unarmed control virus (FIG.25).

Experiment 7 (Anti-Tumor Efficacy of C5a-Armed Adenovirus)

To assess the anti-tumor potency of C5a in the context of non-cytolytictumor infection, we treated established B16-OVA tumors in C57BL/6 miceon days 0, 2 and 4 with either PBS, C5a-expressing- or with unarmedcontrol viruses. Results reveal strong anti-tumor effect by theC5a-expressing virus (FIG. 26).

Experiment 8 (Increased Anti-Tumor T Cell Expansion by C5a-Virus)

To assess whether the observed increase in anti-tumor efficacy ofC5a-expressing virus (FIG. 24) was related to T cells, tumors wereanalyzed by flow cytometry for ovalbumin-specific CD8+ T cells, detectedby staining with APC-conjugated pentamer specific for TCR recognizingMEC I loaded with immunodominant ovalbumin peptide SIINFEKL (ProImmune,USA). Indeed, tumors in the C5a-virus group contained a significantlygreater fraction of tumor-specific CD8 T cells than tumors injected withcontrol virus or PBS (FIG. 27).

Experiment 9 (Transgene Expression From TNF-□-Encoding Adenovirus)

Similar to the C5a virus (FIG. 24 and Experiment 5), we tested theability of hTNF-α-expressing oncolytic adenovirus to mediate secretionof the transgene of choice. Results confirm expression (FIG. 18).

Experiment 10 (Biological Effect of Expressed Transgene is Retained inOncolytic Adenovirus)

In order to assess whether the adenovirus-expressed transgene retainsits biological effects, virus-free (100 kD-filtered) supernatant fromA549 cells infected with control unarmed virus or withTNF-alpha-expressing virus (varying VPs/cell, 72 h p.i.) was appliedonto WEHI-13VAR (ATCC CRL-2148) cells, which are sensitive to TNF-alpha,and these cells were assessed for viability 72 hours after exposure tothe supernatant. (For example Espevik T et al. J Immunol Methods. 1986;95(1): 99-105 describes the method.) Results reveal that TNF-alphaexpressed from oncolytic adenovirus retains potent biological effects(FIG. 19).

Experiment 11 (Oncolytic Cytokine-Expressing Viruses Retain Cell-KillingAbility In Vitro)

Because TNF-alpha may have antiviral effects, it was important toconfirm that oncolytic effect of adenovirus expressing TNF-α retains itsability to infect and kill cancer cells. Several cancer cell lines inculture were infected with TNF-alpha-expressing or with control virusesand assessed for viability by CelltiterGlo AQ MTS assay, as permanufacturer's instructions (Promega, USA). Results show the virus isoncolytic in vitro (FIGS. 19-20).

Experiment 12 (Synergy Between Radiotherapy and Oncolytic VirusExpressing TNF Alpha)

We treated nude mice carrying subcutaneous A549 xenograftsintratumorally with viruses with or without concomitant focused externalbeam radiation (XRT) (FIG. 21). RD indicates replication deficient virusand unarmed virus is an oncolytic virus without TNFalpha.

Experiment 13 (Increased Anti-Tumor Efficacy of TNF-Alpha-ExpressingAdenovirus in Immunocompetent Hosts)

To test whether oncolytic adenovirus, which does not replicate in murinecells, might still be able to cause anti-tumor effects in vivo inimmunocompetent mice, mice with established B16.OVA tumors were injectedintratumorally with TNF-alpha-expressing or unarmed control virus orPBS, in a manner similar to as in FIG. 26. Results show greater overalltumor control with TNF-alpha-expressing virus compared to controls (FIG.22), suggesting that human TNF-alpha is partially active in mice andsupporting the notion of arming viruses to achieve greater anti-tumoreffects.

Experiment 14 (Increased Anti-Tumor T Cell Expansion by TNFα-Virus)

Similar to Experiment 11, we wanted to test whether the observedanti-tumor effect of the TNF-alpha-expressing virus was associated withinduction of tumor-specific cytolytic T cell responses. We extractedtumors and processed them for flow cytometric analysis, as in Experiment11. Results (FIG. 23) indeed confirm that also TNF-alpha expressionfacilitates expansion of tumor-specific T cells at the tumor site,strongly arguing in favor of the proposed technology.

FIGS. 28-32, 36 and 48 illustrate the methods and mechanisms of thepresent invention.

Experiment 15 (Combination Experiment With Two Different AdenoviralVectors and OT1(Ad-mTNFa/Ad-mIL2+OT1)):

Model:

C57BL/6 with B16-OVA (0.25×10e6 cells per animal)

Groups:

Ad5-CMV-mTNFa (1×10e9 VP)

Ad5-CMV-mIL2 (1×10e9 VP)

Ad5-CMV-mTNFa+Ad5-CMV-mIL2 (0.5+0.5×10e9 VP)

Ad5-CMV-mTNFa+OT1

Ad5-CMV-mIL2+OT1

Ad5-CMV-mTNFa+Ad5-CMV-mIL2+OT1

Ad5Luc1+OT1

No injection (mock-mock)

Ad5 vector is a vector of non-replicative human adenovirus coding for amouse transgene. The constructs were made with AdEasy technology(Agilent Inc); the transgene cassette (driven by a CMV promoter) is inthe deleted E1 region (see e.g. Diaconu I et al. Cancer Res. 2012 May 1;72(9):2327-38).

Group size:

n=9

100 ordered

Treatment schedule:

OT1 cells: 1.5×10e6 per animal i.p. on Day 1 (same amount as in previousexperiment, not 2×10e6)

Virus injections: for single agents: 1×10e9 virus particles (OD260) onDay 1 and weekly thereafter; for combination: 0.5×10e9 VP+0.5×10e9 VP onDay 1 and weekly thereafter

Endpoint: Tumor volume (measured every 2 days for the first week andthen every 3 days)

Further Experiments Supporting the Invention

Several animal experiments support the invention. First we screenedoptimal cytokine candidates to combine with adoptive T-cell transferusing recombinant murine forms of cytokines (FIG. 49). A cytokine(s)is(are) selected from the following group: interferon alpha, interferonbeta, interferon gamma, complement C5a, GMCSF, IL-2, TNFalpha, CD40L,IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2,CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2,CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26,CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9,CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1,CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3,CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4,CXCR5, CXCR6, CXCR7 and XCL2. A schematic of the general layout of thevirus genome comprising the cytokine transgene or two transgenes areshown in FIGS. 33 and 34. FIG. 35 shows nucleotide and amino acidsequences of 2A. Nucleotide sequences of the viral vectors comprisingtransgenes C5a, hCD40L, hIFNa2, hIFNb1, hIFNg1, hIL2 or TNFa are shownin SEQ ID NOs 1-7, respectively (Ad5/3-E2F-D24-transgene). Furthermore,nucleotide sequences of the viral vectors comprising two transgenes, theother one being IL-2 and the other one C5a, CD40L, IFNa2, IFNb, IFNg,GMCSF or TNFa, are shown in SEQ ID NOs 8-21 (SEQ ID NO: 8 C5a-2A-IL2,SEQ ID NO: 9 IFNa-2A-IL2, SEQ ID NO: 10 TNFa-2A-IL2, SEQ ID NO: 11CD40L-2A-IL2, SEQ ID NO: 12 IFNb-2A-IL2, SEQ ID NO: 13 GMCSF-2A-IL2, SEQID NO: 14 IFNg-2A-IL2, SEQ ID NO: 15 C5a-IRES-IL2, SEQ ID NO: 16IFNa-IRES-IL2, SEQ ID NO: 17 TNFa-IRES-IL2, SEQ ID NO: 18CD40L-IRES-IL2, SEQ ID NO: 19 IFNb-IRES-IL2, SEQ ID NO: 20GMCSF-IRES-IL2, SEQ ID NO: 21 IFNg-IRES-IL2)(Ad5/3-E2F-D24-transgene-IRES/2A-transgene).

Several of the best candidates were chosen for a cytokine/viruscombination experiment, where regimen roughly stay the same and all themice receive intraperitoneal injection of CD8a+ enriched OT-Ilymphocytes and intratumoral treatments of chosen cytokine mixed withadenovirus. In addition, a separate trafficking experiment was conductedusing our existing replication deficient adenoviruses coding for eithermouse cytokines or human cytokines with proven activity in mice (FIG.50). RD indicates replication deficient virus. Based on theseexperiments a final cytokine candidate or candidates can be chosen andanalyzed further, even in the clinic.

Results of the experiments indicate that a) virus injection into tumorsresults in enhanced trafficking of T-cell to the tumor, b) virusinjection results in enhanced MHC1 expression in tumors, c) dangersignaling is activated resulting in less tolerance andimmunosuppression, d) T-cells propagate at the tumor following virusinjections. Importantly, adding a cytokine as a transgene enhanced eachof these effects. Of note, dual transgenes enhanced the effect further.Thus, intratumoral injection of cytokine armed oncolytic adenovirusenhanced the effect of adoptive cell transfer in a synergistic manner,over what could be achieved with either virus vectors or adoptive celltransfer alone.

To study T cell trafficking and biodistribution after adoptive transfer,a SPECT/CT imaging experiment was conducted (FIG. 51). CD8a+ enrichedOT-I lymphocytes were radiolabeled with 111In and adoptively transferredinto recipient mice.

Since the half-life of indium oxine is relatively short (2,83 days), themaximum surveillance period for the imaging was limited to 7 days. Dueto this restriction, cells were labeled in two batches and transferredinto mice at two different time points. The imaging data from the firstbatch covers trafficking events from days 0-7, whereas the second batchenables us to observe events in tumors during days 8-14 post-virus.

Oncolytic Ad3 Viruses (FIGS. 37-40, SEQ ID NOs 30 and 31(Ad3-hTERT-E3del-CMV-CD40L and Ad3-hTERT-E3del-E2F-CD40L))

Cloning strategy:

1. Construction of Ad3 3′ end plasmid containing correspondingexpression cassette, this plasmid contains 3′ITR of Ad3 genome, the E3region from 29,892 to 30,947 of the Ad3 genome were replaced by theexpression cassette. (Note: We take advantage of EcoRI restriction sitein the Ad3 genome close to 3′ end)

2. Construction of Ad3 5′ end plasmid, this plasmid contains 5′ITR andhTERT-E1. (Note: We take advantage of unique restriction site NotI inAd3 genome and Nhel restriction site close to 5′ end)

3. Construction of pWEA-Ad3-hTERT-CMV-CD40L and pWEA-Ad3-hTERT-E2F-CD40L(Note: We take advantage of phage packaging system)

Construction of Ad3 3′ end plasmid containing corresponding expressioncassette:

1. PCR amplify E2F promoter, forward primer:5′AAAttaattaatggtaccatccggacaaagc3′ (SEQ ID NO: 22), reverse primer5′TTTgctagcggcgagggctcgatcc3′ (SEQ ID NO: 23). Cloned into TA vectorpGEM-T (promega)→pGemT-E2F

2. PCR amplify CD40L fragment, forward primer:5′TAGCTGCTAGCATGATCGAAACATACAAC3′ (SEQ ID NO: 26), reverse primer:5′GTCAATTTGGGCCCTCAGAGTTTGAGTAAGCCAA3′ (SEQ ID NO: 27). Cloned intopGEM-T→pGemT-CD40L.

3. Our Ad3 3′ end plasmid containing CMV-GFP(pWEA-Ad3-3′ end-CMVGFP) wasdigested with NheI/ApaI to remove GFP, pGemT-CD40L was digested withNheI/ApaI→pWEA-Ad3-3′ end-CMV-CD40L.

4. The CMV promoter in pWEA-Ad3-3′ end-CMV-CD40L was replaced by E2Fpromoter (pGemT-E2F was digested with PacI/NheI)→pWEA-Ad3-3′end-E2F-CD40L

Construction of Ad3 5′ end plasmid containing hTERT-E1:

1. PCR amplify 5′ end of Ad3 genome from pKBS2-hTERT (plasmid fromAd3-hTERT-E1A paper), forward primer5′gtcagtttaaacttaggccggccctatctatataatataccttatagatggaatgg3′ (SEQ ID NO:28), reverse primer 5′CTTCATCAGCAGCTAGCAGCATAGAATCAG3′ (SEQ ID NO: 29).Cloned into pGem-T→pGemT-Ad3-5′ end-hTERT.

2. Plasmid pWEA-Ad3 (which contains the whole ad3 genome) was digestedwith FseI/NotI, the 13.2 kb fragment that contains the 5′ end of Ad3genome was cloned into a vector modified from pBluescript KS(-) (therestriction sites between SacI and XbaI were modified asSacI-PmeI-MluI-FseI-SalI-NotI-XbaI)→pBS-Ad3-5′ end

3. Plasmid pBS-Ad3-5′ end was digested with PmeI/NheI, the ˜800 bpfragment that contains 5′ITR were replaced by the correspondingPmeI/Nhel fragment from pGemT-Ad3-5′ end-hTERT→pBS-Ad3-5′ end-hTERT

pWEA-Ad3-hTERT-CMV-CD40L and pWEA-Ad3-hTERT-E2F-CD40L:

1. Plasmid pWEA-Ad3-hTERT-E2F- was digested with EcoRI to remove the 3′end genome, and ligate with the corresponding fragment containingexpression cassette from pWEA-Ad3-3′ end-CMV-, pWEA-Ad3-3′ end-CMV-CD40Land pWEA-Ad3-3′ end-E2F-CD40L

2. The ligation were packaged into phages using Gigapack III pluspackaging extract (Stratagen) and propagated (X11 blue strain)

The functionality of Ad3 viruses were tested in vitro and the resultsare shown in FIG. 41. All new viruses were functional and capable ofinfecting tumour cell lines.

The viruses were also tested on CHO-K7 but they showed no effect on theviability of these cells during the TCID₅₀. This was probably due to thelack of human-like desmoglein-2 on the surface of these hamster cells.

In Vivo Results of AD3 Vectors

All animal experiments were approved by the Experimental AnimalCommittee of the University of Helsinki and the Provincial Government ofSouthern Finland. Mice were frequently monitored for their health statusand euthanized as soon as signs for pain or distress was noticed. Femalefox chase severe combined immunodeficiency mice (Charles River) wereused.

An orthotopic model of peritoneally disseminated ovarian cancer wasdeveloped by injecting 5×10e6 SKOV3-luc cells intraperitoneally in 300ml of pure Dulbecco's modified Eagle's medium into severe combinedimmunodeficiency mice (n=5 per group). After 3 days mice were imagednon-invasively and treated intraperitoneally by injecting PBS or 109 VPin

PBS per mouse. The mice were imaged on day 3, 7, 14, 21 and 25 usingIVIS 100 (Xenogen, Alameda, Calif.) to estimate the number of tumorcells in the mice. For bioluminescence imaging, 150 mg/kg D-luciferin(Promega) was injected intraperitoneally and captured 10 min later with10 s exposure time, if/stop, medium binning and open filter. Duringimaging the mice were in isoflurane gas anesthesia. Images were overlaidwith Living Image 2.50 (Xenogen). Total flux (photons/s) was measured bydrawing regions of interest around the peritoneal area of the mice.Background was subtracted.

FIG. 45 shows anti-tumor efficacy of Ad3 based viruses in vivo.

MTS Cell Proliferation Assay (FIGS. 42-44)

On day one, 105 cells per well (A549, PC3-MM2 or SKOV3-luc) were seededinto 96-well plates in 100 μl of growth medium (GM), which contained 5%of FBS. On day two, the monolayer was washed once with GM containing 5%of FBS. Then the cells were infected with different viruses at doses of100, 10, 1, 0.1 and 0 virus particles per cell. Thereafter the cellswere incubated for one hour on a rocking machine and then washed withGM. After adding new 5% GM the cells were left to the incubator and theGM was replaced every fourth day. The test was terminated by adding mtsreagent (Promega) after the cytopathic effect of one of the testedviruses reached 100% with the highest concentration. After two hours ofincubation the absorbance was measured at 490 nm filter. The backgroundwas then subtracted and results analyzed.

Therapeutic Window of Oncolytic Adenovirus Coding for Murine CD40L inImmunocompetent Mice

In immunocompetent animals, viral genomes are present in tumors afteri.v. injections (FIG. 46). Albino C57 mice were inoculated s.c. withmouse B16-ova cells and treated intravenously with 5 different viraldoses of Ad5 based virus coding for mouse CD40L (see experiments 4 and15). Tumors of 3 animals per group were collected and stored at -80° C.Total DNA was extracted and viral DNA load was studied with quantitativePCR. Viral E4 copy numbers were normalized to genomic DNA with mouseB-actin primers. In FIG. 46 each icon represents one tumor; horizontalline indicates the median of the group. Mock: n=5; Dose 5: n=4; Dose 4:n=4; Dose 3: n=4; Dose 2: n=6; Dose 1: n=2; Dose 2 i.t.: n=4. DOSE 5:1×10¹¹ VP/mouse; DOSE 4: 3×10¹⁰ VP/mouse; DOSE 3: 1×10¹⁰ VP/mouse; DOSE2: 1×10⁹ VP/mouse; DOSE 1: 1×10⁸ VP/mouse; Positive control (DOSE 2intratumorally.)

With dose 5.67% of mice had signs of liver toxicity. Dose 4 was able toachieve good tumor transduction following i.v. delivery, without signsof liver toxicity.

Results of the liver enzyme release experiment are shown in FIG. 47.Liver enzyme release experiment was carried out as follows. All animalprotocols were reviewed and approved by the Experimental AnimalCommittee of the University of Helsinki and the Provincial Government ofSouthern Finland. Three- to four-week-old female albino C57 mice (HarlanLaboratories, The Netherlands) were injected with 2.5×10⁵ B16-ova cellssubcutaneously in both flanks and randomized into 7 groups (3mice/group). Ad5/3 CMV-mCD40L virus diluted in phosphate buffered saline(PBS) was injected intravenously at 10⁸-10¹¹ viral particles (VP)/mouse(dose 1-dose 5). One treatment group received dose 2 (10⁹ VP/cell)intratumorally as positive control. Animals were anesthetized prior toany procedures and the health status monitored daily. 48h post virusinjection the mice were sacrificed and the blood was collected bycardiac puncture. Serum was separated by centrifuging blood samples at5000 rpm for 10 minutes. Alanine aminotransferase (ALT) and aspartateaminotransferase (AST) levels (Units/litre) in serum samples werequantified at University of Helsinki Clinical Chemistry Core using theSiemens ADVIA 1650 clinical chemistry analyzer. Hemolytic samples wereexcluded from the analysis, as serum hemolysis may interfere with theassays (false high ALT and AST levels). The bars show averages+SEM.

REFERENCES

Blair G E, Dixon S C, Griffiths S A, Zajdel M E. Restricted replicationof human adenovirus type 5 in mouse cell lines. Virus Res. 1989December; 14(4):339-46.

Ekkens M J, Shedlock D J, Jung E, Troy A, Pearce E L, Shen H, Pearce EJ. Th1 and Th2 cells help CD8 T-cell responses. Infect Immun. 2007 May;75(5):2291-6.

Kratky W, Reis e Sousa C, Oxenius A, Spörri R. Direct activation ofantigen-presenting cells is required for CD8+ T-cell priming and tumorvaccination. Proc Natl Acad Sci USA. 2011 Oct. 18; 108(42):17414-9.

Lugade A A, Sorensen E W, Gerber S A, Moran J P, Frelinger J G, Lord EM. Radiation-induced IFN-gamma production within the tumormicroenvironment influences antitumor immunity. J Immunol. 2008 Mar. 1;180(5):3132-9.

Propper D J, Chao D, Braybrooke J P, Bahl P, Thavasu P, Balkwill F,Turley H, Dobbs N, Gatter K, Talbot D C, Harris A L, Ganesan T S.Low-dose IFN-gamma induces tumor MHC expression in metastatic malignantmelanoma. Clin Cancer Res. 2003 January ; 9(1):84-92.

Schroder K, Hertzog P J, Ravasi T, Hume D A. Interferon-gamma: anoverview of signals, mechanisms and functions. J Leukoc Biol. 2004February; 75(2):163-89.

Street D, Kaufmann A M, Vaughan A, Fisher S G, Hunter M, SchreckenbergerC, Potkul R K, Gissmann L, Qiao L. Interferon-gamma enhancessusceptibility of cervical cancer cells to lysis by tumor-specificcytotoxic T cells. Gynecol Oncol. 1997 May; 65(2):265-72.

References for Viral Constructs

Blair G E, Dixon S C, Griffiths S A, Zajdel M E. Restricted replicationof human adenovirus type 5 in mouse cell lines. Virus Res. 1989December; 14(4):339-46.

Ekkens M J, Shedlock D J, Jung E, Troy A, Pearce E L, Shen H, Pearce EJ. Th1 and Th2 cells help CD8 T-cell responses. Infect Immun. 2007 May;75(5):2291-6.

Kratky W, Reis e Sousa C, Oxenius A, Spörri R. Direct activation ofantigen-presenting cells is required for CD8+ T-cell priming and tumorvaccination. Proc Natl Acad Sci U S A. 2011 Oct. 18; 108(42):17414-9.

Lugade A A, Sorensen E W, Gerber S A, Moran J P, Frelinger J G, Lord EM. Radiation-induced IFN-gamma production within the tumormicroenvironment influences antitumor immunity. J Immunol. 2008 Mar. 1;180(5):3132-9.

Propper D J, Chao D, Braybrooke J P, Bahl P, Thavasu P, Balkwill F,Turley H, Dobbs N, Gatter K, Talbot D C, Harris A L, Ganesan T S.Low-dose IFN-gamma induces tumor MHC expression in metastatic malignantmelanoma. Clin Cancer Res. 2003 January; 9(1):84-92.

Schroder K, Hertzog P J, Ravasi T, Hume DA. Interferon-gamma: anoverview of signals, mechanisms and functions. J Leukoc Biol. 2004February; 75(2):163-89.

Street D, Kaufmann A M, Vaughan A, Fisher S G, Hunter M, SchreckenbergerC, Potkul R K, Gissmann L, Qiao L. Interferon-gamma enhancessusceptibility of cervical cancer cells to lysis by tumor-specificcytotoxic T cells. Gynecol Oncol. 1997 May; 65(2):265-72.

1. An oncolytic adenoviral vector comprising: i) an adenovirus serotype5 (Ad5) nucleic acid backbone comprising a 5/3 chimeric fiber knob: ii)E2F1 promoter for tumor specific expression of E1A; iii) a 24 bpdeletion (D24) in the Rb binding constant region 2 of adenoviral E1; iv)a nucleic acid sequence deletion of viral gp19k and 6.7k reading frames;and v) a nucleic acid sequence encoding at least one cytokine transgenein the place of the deleted gp19k/6.7K in the E3 region resulting inreplication-associated control of transgene expression under the viralE3 promoter, wherein the cytokine is selected from a group consisting ofinterferon alpha, interferon beta, interferon gamma, complement C5a,IL-2, TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12,CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18,CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24,CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5(=RANTES), CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8,CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13,CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.
 2. Theoncolytic adenoviral vector according to claim 1, wherein the vectorcodes for two or more cytokines.
 3. The oncolytic adenoviral vectoraccording to claim 2, wherein the vector comprises an internal ribosomalentry site (IRES) or a ribosome shunt site 2A between the twotransgenes.
 4. A serotype 3 (Ad3) oncolytic adenoviral vectorcomprising: a deletion in the E3 area affecting E3 9 kDa, E3 10.2 kDa,E3 15.2 kDa and E3 15.3 kDa, and an exogenous or tumor specific promoterfor expression of a transgene in the place of the deleted area of E3. 5.The oncolytic adenoviral vector according to claim 4, wherein saidtransgene is a cytokine selected from a group consisting of interferonalpha, interferon beta, interferon gamma, complement C5a, IL-2,TNFalpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13,CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19,CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1,CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5(=RANTES), CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8,CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13,CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.
 6. Theoncolytic adenoviral vector according to claim 4, wherein the vectorcodes for two or more cytokines.
 7. The oncolytic adenoviral vectoraccording to claim 6, wherein the vector comprises an internal ribosomalentry site (IRES) or a ribosome shunt site 2A between the twotransgenes.
 8. A pharmaceutical kit comprising an adoptive celltherapeutic composition and an oncolytic adenoviral vector according toany one of claims 1-7, wherein the adoptive cell therapeutic compositionis formulated in a first formulation and the oncolytic adenoviral vectorcoding for at least one cytokine is formulated in a second formulation.9. The pharmaceutical kit according to claim 8, wherein the first andthe second formulations are for simultaneous or sequentialadministration, in any order, to a subject.
 10. The kit according toclaim 8, wherein the adoptive cell therapeutic composition comprises acell type selected from a group consisting of a tumor infiltratinglymphocyte (TIL), T-cell receptor modified lymphocytes and chimericantigen receptor modified lymphocytes.
 11. The kit according to claim 8,wherein the adoptive cell therapeutic composition comprises a cell typeselected from a group consisting of T-cells, CD8+ cells, CD4+ cells,NK-cells, delta-gamma T-cells, regulatory T-cells, and peripheral bloodmononuclear cells.
 12. A pharmaceutical composition comprising anoncolytic vector according to claim
 1. 13. A pharmaceutical compositioncomprising an oncolytic vector according to claim 4.